The objective of this proposal is to identify the direct measurement the enteric sensory, modulatory and motor neurotransmitters that regulate athe peristaltic reflex elicited by muscle stretch and mucosal stimulation in rat colon and human intestine. Our previous studies and preliminary experiments have led us to identify neuronal circuits consisting of modulatory neurons coupled to motor neurons that account for reciprocal circular and longitudinal muscle activity during the ascending and descending phases of the reflex. The experimental approach exploits novel preparations that enable identification of various components of the circuits: (i) flat-sheet compartmented intestinal segments with which to measure specifically the release of sensory transmitters; (ii) longitudinal muscle strips with adherent myenteric plexus with which to identify the coupling of modulatory neurons to motor neurons innervating longitudinal muscle; (iii) circular muscle st rips devoid of myenteric plexus with which to measure presynaptic regulation of transmitter release, and from which to prepare synaptosomes to characterize the binding of transmitters to presynaptic receptors; and (iv) freshly dispersed myenteric ganglia with which to examine feedback regulation of modulatory interneurons within the plexus. In Specific Aim I, studies in rat colonic and human intestinal segments will identify (a) the role of an integrated modulatory circuit consisting of somatostatin, enkephalin and GABA neurons that regulates motor neurons innervating circular and longitudinal muscle during the ascending and descending phases of the reflex, and (b) the role of 5-HT acting via neuronal 5-HT4 receptors in regulating release of the sensory transmitters, CGRP and the tachykinins, substance P (SP) and neurokinin A (NKA). In Specific Aim II, studies in myenteric plexus-longitudinal muscle strips will demonstrate that the modulatory circuit acts via VIP/PACAP/NOS neurons to regulate cholinergic/tachykinin motor neurons that innervate longitudinal muscle. In Specific Aim III, studies on circular muscle strips containing only axonal terminals will identify the presence of regulatory presynaptic (i.e., axo-axonal) interactions between modulatory nerve terminals and excitatory and inhibitory motor nerve terminals. The existence of these interactions will be corroborated by radioligand binding studies on synaptosomes derived from these terminals. In Specific Aim IV, studies on isolated ganglia devoid of axonal projections to muscle will characterize the interplay of modulatory interneurons and demonstrate the role of somatostatin interneurons as the neuronal switch between the two phases of the reflex. The combined approach embodied by these aims should advance our understanding of a major neuromuscular function.